1. Field of the Invention
The present invention relates to a method of analyzing methylated DNA. The present invention relates in particular to a method of analyzing methylated DNA, a primer set, a method of judging a reliability of a step of concentrating methylated DNA, a method of detecting non-methylated DNA in a methylated DNA concentrate, a method of calculating a concentration rate of methylated DNA, and a method of evaluating a purity of a methylated DNA concentrate.
2. Description of the Related Art
Methylation of a CpG site in DNA significantly influences gene expression. For example, abnormalities in methylation of DNA are known to participate in the onset of diseases, for example, in the expression of genes related to malignant transformation of cells. Accordingly, the methylation of DNA in various genes is comprehensively studied in various genes in order to develop diagnostic and therapeutic methods for diseases such as cancer.
In analysis of the methylation of DNA, methylated DNA is concentrated for example by immunoprecipitation of methylated DNA with an anti-methylated cytosine antibody, an anti-methylated cytidine antibody or a methylated DNA-binding protein. Thereafter, profiling of DNA contained in the resulting concentrate is conducted. In analysis of such DNA methylation, the efficiency of analysis may be decreased when non-methylated DNA is contained in the methylated DNA concentrate. For example, in the immunoprecipitation of methylated DNA, non-methylated DNA may be bound or adsorbed nonspecifically to an anti-methylated cytosine antibody, an anti-methylated cytidine antibody, a methylated DNA-binding protein, or bead. Consequently, the presence or absence, or the content, of non-methylated DNA in the methylated DNA concentrate is confirmed in such analysis of DNA methylation.
Detection of non-methylated DNA in a methylated DNA concentrate is conducted by using, as an indicator, a housekeeping gene that has been estimated to be non-methylated. In a method described in Ilana Keshet. et al., Nature Genetics, February 2006; 38(2): 149-53, a glyceraldehyde-3-phosphate dehydrogenase (hereinafter referred to as “GAPDH”) gene is used as an indicator of non-methylated DNA. In this method, an amount of a methylated DNA concentrate concentrated by immunoprecipitation of methylated DNA is determined by standardization with the detected GAPDH gene. In a method described in Hayashi H. et al., Human Genetics, January 2007; 120(5):701-11, a GAPDH gene and β-actin gene are used as non-methylated DNAs. In the method described in this Non-Patent Document 2, the concentration rate of methylated DNA is evaluated by using, as an indicator, the absence of detection of the GAPDH gene and β-actin gene in the methylated DNA concentrate obtained by immunoprecipitation of methylated DNA.
However, there are cases where cytosine at a CpG site in each of the GAPDH gene and β-actin gene has been actually methylated. It follows that when housekeeping genes such as GAPDH gene and β-actin gene are used as the indicator, non-methylated DNA in a methylated DNA concentrate may not be accurately detected. Accordingly, there are cases where evaluation of the concentration rate and purity of a methylated DNA concentrate, or judgment of the reliability of its concentrating step, cannot be accurately conducted.
The state of the housekeeping gene used as an indicator can be confirmed by determining its DNA sequence after treatment with hydrogen sulfite, in order to secure the accuracy of the concentration rate and the like of a methylated DNA concentrate. In this case, however, the operation of confirming the state of methylation is troublesome and time-consuming. As a result, there is a problem that methylated DNA cannot be efficiently and easily analyzed by using a methylated DNA concentrate.